The mammalian polo-like kinase, Plk1, is essential for cell division, apparently required at several points in mitosis and cytokinesis. Because they act at several steps during cell division, genetic data on the M-phase polo-like kinases must be complemented with biochemical analysis. In order to describe how Plk1 executes its various functions, characterization of its substrates and their functions is required. We have three specific aims: i) Plk1 depletion in tumor and normal cells. When expression of Plk1 is silenced by RNA interference (RNAi), tumor cells undergo p53- independent apoptosis. We propose to determine the molecular events that lead to cell death in tumor cells depleted of Plk1. Because Plk1 may be regarded as a potential target in cancer therapy, comparison of normal cells to tumor cells in this regard is necessary. Normal cells may be resistant to cell death as a consequence of checkpoint controls that tumor cells lack. Plk1 expression in normal cells will be silenced using lentiviruses to express various RNAis that target different sequences in Plk1 mRNA. We shall determine if this causes normal cells to undergo apoptosis and whether or not it is p53-dependent. Plk1 hypomorphs will be selected from cells expressing RNAi targeted to different Plk1 mRNA sequences and examined for centrosome deficiencies, spindle defects, and development of aneuploidy, which are all relevant to tumor development, ii) Plk1 substrates. We have identified several Plk1 substrates that appear to have important functions in cell division and that are required for cell viability. We propose to use RNAi to deplete these substrates and to mutate phosphorylation sites in these substrates to develop a more complete picture of their role and that of Plk1 in cell division, iii) MKLP-interactive proteins. One Plk1 substrate is the mitotic kinesin-like protein (MKLP1), which we have shown is essential for cytokinesis but not karyokinesis. We will use techniques previously successful for Plk1 to identify MKLP1- interacting proteins that may contribute to MKLP1 functions during cytokinesis. We shall study interacting proteins in vitro and in vivo with wild type MKLP1 and MKLP1 with Plk1 phosphorylation site mutations. The role of MKLPl-interacting proteins that may promote cytokinesis will be also investigated with RNAi methods.